Information on cytokines and cytology obtained from biological specimens are combined in methods of predicting the risk that dysplasia will progress to cancer.
1. Screening for Dysplasia
A well known problem in clinical medicine is that, although precancerous lesions (dysplasias) are relatively common, only a small proportion of these lesions progress to actual cancer. In the absence of a reliable means to differentiate between lesions that are likely to progress to cancer and those that are not, clinicians generally take a conservative approach and aggressively treat all lesions that exceed some threshold of abnormality. One disadvantage of such a conservative approach is that a significant fraction of these patients receive unnecessary and/or excessively aggressive treatments, many of which are known to produce serious side effects. A second disadvantage is that such unnecessary treatments consume large amounts of limited medical resources, thereby diverting these resources from other patients who could derive more benefit from them. Thus there is a need for a means to differentiate between precancerous lesions that are likely to progress to cancer and those that are not.
Disease screening programs present a challenge in that the tests used must be capable of quickly, accurately and efficiently examining large numbers of asymptomatic individuals in order to detect those few who exhibit subclinical indications of having the disease or a precursor condition. In other words, disease screening is an exercise in rare event detection in which the test used must be sensitive enough to detect subclinical manifestations of the disease and specific enough to accurately differentiate these manifestations from similar manifestations resulting from other causes. An additional complication, particularly in the area of cancer screening, is that once initiated, the disease does not always progress to the point of becoming symptomatic, but may stop at some point or even regress back to normal.
Cervical cancer screening and the subsequent follow up to abnormal screening results provide what is arguably the best documented example of the success of cancer screening. Similar to most screening tests, cervical cancer screening is performed on large asymptomatic patient populations with the intent of identifying those few members of these populations who could benefit from treatment or other appropriate intervention. Because large numbers of asymptomatic individuals must be screened in order to identify the few that exhibit the target disease state, logistics and economics play crucial roles in the deployment and operation of screening programs In particular, the large number of individuals to be screened dictates that the logistics of sample collection and testing be highly efficient. The high costs associated with the confirmation of an abnormal screening result and the even higher costs of treatment (if the abnormal result is confirmed) also impose stringent requirements on the screening process including that the screening test be highly specific and that the costs of screening be minimized.
Since the mid-1940's, cervical cancer screening has been performed using the Pap test, in which cells collected from each individual are examined cytologically to identify those samples that contain cells that exhibit the morphological abnormalities that indicate the presence of dysplasia or cancer. Patients from whom cytologically abnormal specimens are obtained receive follow up, typically by the histological examination of tissue samples obtained by biopsy, in order to confirm the presence of the abnormality and, if confirmed, to diagnose the specific disease state that is present. This diagnosis, in turn, provides the basis for treatment planning and delivery.
At present, cervical cancer screening is based upon the morphological evaluation of squamous epithelial cells and, in some cases, endocervical cells, obtained from the cervix. In the original form of this test these were cells that had been exfoliated from the cervix, but since the late 1940's they have been obtained by scraping the cervix with a spatula, brush or broom device. The major reason for this change in sample collection method was to obtain a “cleaner” sample that is enriched in the cervical squamous epithelial cells to be morphologically evaluated. Until the early 1990's these samples were typically smeared onto a microscope slide in preparation for cytological evaluation. Although this slide preparation method is simple and effective, the resulting specimens often contain clusters and clumps of cells, mucus, bacteria, fungi, yeasts, and non-epithelial cells that can impair the examination of the epithelial cells of clinical interest.
In the late 1990's smears began to be displaced by “monolayer” (or “liquid based”) preparations that facilitate specimen evaluation by better dispersing the epithelial cells, while eliminating or significantly reducing the amount of mucus and the numbers of non-epithelial cells on the slide by means of a purification step in the slide preparation procedure. At the present time these monolayer preparations, which are endorsed by medical societies and National Health Authorities worldwide, account for over 80% of the cervical screening specimens in the US and UK, and comprise a significant, and growing, fraction of the cervical screening specimens worldwide.
Statistics published by the World Health Organization indicate that the incidence rate of cervical cancer varies by country and is generally in the range of 0.02 to 0.1%. The Pap test is by far the predominant method used in cervical cancer screening worldwide. In this test cells squamous epithelial cells collected from the cervix are cytologically examined, and any morphological abnormalities observed in these cells are classified in accordance with internationally accepted criteria (the Bethesda criteria). These categories are:
(a) Within Normal Limits (WNL): no significant abnormalities noted
(b) Atypical Squamous Cells of Undetermined Significance (ASCUS): a category used primarily in the US for cells exhibiting relatively minor morphological abnormalities that do not fall into the other classifications.
(c) Low Grade Squamous Interepithelial Lesion (LSIL or LGSIL): moderate morphological abnormalities consistent with dysplasia. Cells exhibiting morphological changes consistent with viral infection may also be included in this category.
(d) High Grade Squamous Interepithelial Lesion (HSIL or HGSIL): severe dysplastic morphological abnormalities. This is the level at which standards of care generally prescribe aggressive medical intervention.
(e) Cancer.
Although the numbers vary somewhat by patient population, a realistic approximation is that in developed countries, the outcome of a cervical cancer screening program will consist of approximately 90% WNL; 9% ASCUS+LSIL; 0.9% HSIL and 0.1% cancer. These numbers indicate that only a small percentage of cervical dysplasias actually progress to cervical cancer. This is supported by the well established observation that most dysplasias are spontaneously cleared by the body without the need for therapeutic intervention. A means of reliably predicting which dysplasias are likely to progress to cancer is needed in order to optimally use scarce medical resources. It is also well established that when biopsy is used for confirmation, the sensitivity (% of dysplasias detected)
Specificity is the percent of normals correctly identified as such; the false positive rate is 1-specificity.
From an operational perspective, a sensitivity of 70% means that approximately one third of the individuals in the population being screened who have cervical dysplasia or cervical cancer are not detected by the Pap test. From this it is obvious that there is a need to increase the sensitivity of the test that is used for cervical cancer screening. Similarly, a specificity of 70% means that approximately one third of the cases classified by the test as having dysplasia or cancer cannot be confirmed and are generally reclassified as being WNL. In addition to the emotional effects of such an erroneous classification, these false positive results have a significant economic impact as the follow up testing needed to identify the true positive cases within this group is far more expensive in terms of the money and medical resources required than is the initial screening. Given the severe constraints on the availability of healthcare resources worldwide, expending resources on following up on a false positive screening result means that fewer individuals can be screened and that fewer resources are available for the treatment of individuals who are truly positive for dysplasia or cancer. There is need for improving the specificity of the cervical cancer screening test.
Although the preceding description focused on cervical cancer screening, these and other similar considerations and needs identified apply to screening programs for other cancers such as, but not limited to those of breast, prostate, lung and bladder.
Previous attempts to improve sensitivity and specificity have focused upon the development of assays for the detection of “markers” that can be correlated with the presence of dysplasia, improved methods of morphological evaluation, and the use of surrogate indicators. Markers are cell surface or intracellular molecules whose concentrations significantly increase or decrease if cellular processes are disrupted. The markers that have been used to date have almost exclusively been proteins, but the use of a small number of other types of molecules such as lipids and oligonucleotides has occasionally been explored. Although many of these marker-based tests offer high sensitivity, their specificities tend to be limited due to the fact that these markers are normal constituents of cells and play roles in normal and routine cellular processes. These markers are also not expressed in isolation, but rather as elements of a highly interconnected network of cellular processes wherein the factors causing a change in the expression of one marker can have diverse effects on the expression of many other markers. The networks involved in routine cellular maintenance and repair have proven to be particularly troublesome in this regard. The most fruitful marker-based approaches to date have focused upon correlation of the expression of multiple markers rather than on the expression of a single marker. Limited success has also sometimes been achieved by precisely quantitating the level of marker expression in individual cells. In addition to the numerous technical challenges of making the necessary quantitative measurements, normal inter-individual and even intra-sample variability makes it extremely challenging to determine the true background or reference level for expression of the marker that is needed in order to determine whether a measured change in expression is significant. Similar challenges and limitations apply to the use of improved methods of morphological evaluation, most of which are based upon various methods of automated image analysis. Repairative cellular processes and the need for unusually stringent process control in the preparation and imaging of the specimen are particularly troublesome and limiting in this approach.
The challenges and limitations described above have led to the exploration of the use of surrogate markers for the detection of cancerous and precancerous conditions. Arguably the best developed of these methods is the use of HPV testing for cervical cancer screening. This use is based upon the strong correlation (>90%) that has been observed between infection by one or more “high risk” (oncogenic) strains of the HPV virus, and HSIL/cancer. The argument presented in favor of this approach is that because the HPV virus itself, and the various proteins and other molecules that it produces in cells, are all “foreign”, tests based upon this approach are not subject to many of the limitations outlined above. Although there is a strong correlation between the presence of HPV infection by one or more oncogenic strains and the presence of dysplasias up to and including cancer, it is also well known in the art that only very few such infections actually progress as far as HSIL and even fewer progress to cancer. As a consequence, although HPV tests are highly sensitive, their false positive rates are routinely reported to be in the range of 40-60%. A few reports in the literature have recently suggested that this is a consequence of HPV-induced dysplasias progressing to cancer being not a direct result of the infection itself, but rather being a reflection of relatively rare random errors that can occur during the propagation of the virus within a cell. This, in turn, has led to proposals to use the increased expression of the HPV proteins E6 and E7 that seems to be better correlated with HSIL and cancer as markers. This approach, however, runs into limitations similar to those described above as expression of E6 and E7 is a normal part of the HPV life cycle and is therefore not in and of itself definitive for cells that will progress to cancer.
As long ago as the 1850's it was observed that tumors in some cancer patients who acquired and then recovered from certain life threatening infections, shrank or even completely disappeared. It was also observed that a substantial portion, over 50% in some cases, of the mass of a solid tumor was comprised of white blood cells. More recently it was determined that these tumor-infiltrating lymphocytes (TIL's) consist primarily of T-cells, NK cells, dendritic cells, neutrophils, macrophages and other of the types of cells comprising the innate immune system. Other research has demonstrated that a primary function of these cells is to detect, attack and destroy unneeded, damaged, foreign, infected and otherwise abnormal cells.
2. Relevant Aspects of the Human Immune System
Although the structure of the human immune system, its control, and its relationship with cancer, are not yet completely understood, a few points that pertain to the present invention can be summarized as:
(a) The interactions between the immune system and cancer are complex and not well understood. These interactions can range from the immune system attacking and destroying the cancer to entering a state in which the cancer is tolerated, or even actively promoted. There is also a large and growing body of evidence that a developing cancer can modulate the corresponding immune response by any of a number of means.
(b) Newly appearing dysplastic and cancer cells are initially detected and neutralized or destroyed by effector T-cells, NK-cells, macrophages and other cells of the innate immune system. This is typically described as an inflammatory response on the basis of the types of cytokines that are produced.
(c) A B-cell (antibody, humoral) response to the cancer may be generated.
(d) If the cancer is cleared in a timely manner, the immune response reverts to its resting surveillance state, leaving sensitized memory T- and B-cells that can respond rapidly if another similar cancer is subsequently detected.
(e) If the cancer persists and is not cleared in a timely manner, certain cells of the immune system can undergo phenotypic shifts that reduce or terminate the immune response in the vicinity of the cancer. Among the shifts that have been reported:
Helper T-cells transition from the Th1 phenotype (expressing pro-inflammatory cytokines and promoting an immune response) to the Th2 phenotype (expressing anti-inflammatory cytokines and suppressing the immune response).
A portion of the T-cell population adopts a regulatory (Treg) phenotype that can locally suppress the innate immune response. Less is known about regulatory B-cells (Bregs) that appear to be generated before, or at the same time as Tregs, and locally suppress the immune response.
A portion of the local macrophage population transitions from Type M1 (aggressive) to Type M2 (tolerant).
Th2 helper T-cells, Tregs, M2 macrophages and Bregs secrete anti-inflammatory cytokines that, in addition to locally suppressing the local immune response, can promote angiogenesis and other aspects favorable to tumor proliferation.
Cancer cells can express anti-inflammatory cytokines including, but not limited to, Interleukin-10 (IL-10). Certain of these cytokines, most notably IL-10, down-regulate the cancer-specific immune response by suppressing Interferon Gamma (IFN-γ), IL-2 and IL-12 production. This results in an increased production of other anti-inflammatory cytokines such as IL-4 and IL-6, a reduced display of tumor antigens by the Major Histocompatibility Complex (MHC) on the surfaces of tumor cells, and the inhibition of the presentation of tumor-specific antigens by dendritic and other antigen-presenting cells.
Certain cells of the innate immune system, most notably T-cells and NK cells, individually examine the cell surface markers displayed by the cells comprising the tissues in their vicinity to determine their status. Detection of a cell that displays an abnormal suite of these markers triggers a cytolytic and cytotoxic response from the T- or NK cell that is directed at destroying the abnormal cell. At the same time these cells release a variety cytokines and chemokines that attract macrophages, neutrophils, dendritic cells and other cells of the innate immune system to the site and activate them to continue the destruction of the abnormal cell and to remove the resulting debris. Certain interferons and other molecules that interact directly with the target cell may also be released. The destruction of an abnormal cell therefore usually proceeds rapidly and efficiently, but is in some cases not sufficient to completely eliminate a lesion.
It is reported, primarily from research into the genesis of autoimmune diseases, that an overly prolonged inflammatory immune response of the type described above can result in the activated immune cells damaging and subsequently attacking normal cells in the vicinity of the original lesion. In order to prevent this type of undesirable collateral damage, a highly effective means of terminating the inflammatory immune response is provided and is triggered either when the target cell is destroyed or if the inflammatory response is excessively prolonged. Just as the attack phase of the immune response is largely mediated by pro-inflammatory cytokines, the termination phase is largely mediated by anti-inflammatory cytokines with the balance between them being controlled by a complex network of interactions between the various types of immune cells that are present. Recently research into the use of activated T-, NK and dendritic cells as cancer therapeutic agents has revealed that the balance between pro- and anti-inflammatory cytokines can also be modulated by the target cells themselves. In particular it has been found that in many cases dysplastic and cancerous cells are capable of expressing and releasing sufficient quantities of anti-inflammatory cytokines to suppress or terminate the immune attack upon them and to locally force the innate immune system into a quiescent state Imposition of this local immunosuppressed state is thought to be necessary and sufficient to permit progression of the dysplasia or cancer.